Ribonuclease H (RNase H) plays crucial roles in replication of retroviruses, including the human immunodeficiency virus (HIV) as well as involvement in normal cellular events including DNA replication. The purpose of this project is to gain a detailed understanding of the precise nature of the substrates for RNases H, the chemical makeup of cellular RNases H. The enzymes derived from cellular sources have higher specific activities than those of retroviral origin and have somewhat different specificities. We have found that separation of the RNase H domain of MuLV reverse transcriptase from the polymerase domain creates an active enzyme with specificities different from the parental RNase H but still with low specific activity. If we produce RNases H from yeasts, they have high specific activities regardless of their association with their own special domains. These results indicate that the differences in these proteins reside in the RNase H domain itself.